title: Structural basis for cyclization specificity of two Azotobacter type III polyketide synthases: a single amino acid substitution reverses their cyclization specificity.
abstract: Type III polyketide synthases (PKSs) show diverse cyclization specificity. We previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp-281 of ArsB corresponded to Gly-284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 Å, respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase.
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Satou
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Akimasa
authors.initials: A
authors.lastname: Miyanaga
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Ozawa
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Funa
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Katsuyama
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Ken-Ichi
authors.initials: KI
authors.lastname: Miyazono
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tanokura
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Electronic address: ayasuo@mail.ecc.u-tokyo.ac.jp.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ohnishi
authors.affiliation: Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
authors.forename: Sueharu
authors.initials: S
authors.lastname: Horinouchi
mesh_terms: D019943:Amino Acid Substitution; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D016297:Mutagenesis, Site-Directed; D048630:Polyketide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D061065:Polyketides / Q000138:chemical synthesis* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D061065:Polyketides; D048630:Polyketide Synthases
keywords: Bacterial Metabolism; Crystallography; Kinetics; Polyketides; Site-directed Mutagenesis